Methods for separating frozen-thawed spermatozoa into X-chromosome bearing and Y-chromosome bearing populations

CAFC
  • US 8,652,769 B2
  • Filed: 08/09/2010
  • Issued: 02/18/2014
  • Est. Priority Date: 11/29/2000
  • Status: Active Grant
First Claim
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1. A method of producing a frozen-thawed sorted artificial insemination sample comprising:

  • thawing frozen sperm cells;

    staining said thawed sperm cells with a concentration of a Hoechst 33342 greater than 40 micromolar;

    establishing the staining temperature between about 30°

    C. and about 40 C.°

    ;

    determining a sex characteristic of said sperm cells;

    separating said sperm cells according to the determination of their sex characteristic;

    isolating sperm cells separated according to the determination of their sex characteristic in a collection element at a rate of greater than 1000 sperm per second for either X-chromosome bearing sperm or Y-chromosome bearing sperm;

    establishing a frozen-thawed sorted artificial insemination sample from said sperm cells isolated in said collection element; and

    fertilizing an egg with said frozen-thawed sorted artificial insemination sample at success levels of at least about 70% of the success levels with sperm cells that have not been separated and/or frozen.

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